The present invention relates to a method for increasing stress-resistance to a plant. That is, the method can impart a plant with stress-resistance including good drought resistance and/or resistance to high salt concentration.
The importance of agricultural use of the arid zone, which occupies approximately one third of the land on the earth, is increasingly recognized as a measure against predicted serious food scarcity. This problem should be addressed as soon as possible. Now the proportion of dry and semi-dry soil inappropriate for agriculture is increasing year after year due to saline accumulation, and drying or heat caused by, for example, excessive irrigation water (Manabu Sekiya, et al., Chemical Regulation in Plant Vol. 25, No. 2, 149-162, 1990). One of the solutions to this problem is a method in which resistance mechanisms against these environmental stresses are elucidated and a plant resistant to these stresses is produced.
Plants are immobile. Thus they must be tough enough to endure their environmental changes in order to keep differentiating and growing. Therefore, it is thought that plants have acquired through the course of evolution a response mechanism to respond promptly and adapt to environmental changes. Of the environmental factors surrounding plants, drought and saline accumulation are important factors concerning life or death of terrestrial plants. These factors largely affect plant growth. Plant growth is inhibited by drought stress. That is, it causes decreased turgor pressure and affects various physiological pathways (Shinozaki and Yamaguchi-Shinozaki, Plant Physiol. 115: 327-334, 1997).
In plants, it has been shown that various response mechanisms act against these stresses at an individual level, tissue level, and cellular level, and in addition through molecular biological research at a gene expression level. In other words, in various plants, a response mechanism at a gene expression level, including the presence of many stress-inducible genes whose mRNA amount increases upon drying and treatment with salt, has been elucidated. Plants are thought to acquire resistance from any one of the products of the stress-inducible gene group.
Abscisic acid (ABA), one of a plant""s hormones, is deeply involved in expression of the stress-inducible gene group. When a plant is exposed to a stress, such as drought stress, signal-transduction occurs via ABA dependent pathway and ABA independent pathway, and the signal-transduction regulates the expression of the stress-inducible gene group. The gene group includes those involved in synthesis of compatible solutes, such as proline and glycine betaine. Proline and glycine betaine have been well studied. It is known that a transgenic plant, in which proline or glycine betaine is excessively accumulated by engineering the synthetic or decomposition system, shows resistance to NaCl or low temperature stress.
On the other hands in the biosynthetic pathway for RF0, galactinol is first synthesized by galactinol synthetase. Next, raffinose is synthesized by raffinose synthetase using the galactinol and sucrose as substrates, and finally stachyose is synthesized by stachyose synthetase using the galactinol and raffinose as substrates, as shown in FIG. 10. A generic name for raffinose and stachyose is RF0. So far, every report concerning RF0 suggests that raffinose and stachyose play an important role in drought resistance of seeds (Blackman S. A. et al. Plant Physiol. 100: 225-230, 1992, Ooms J. J. J. et al. Plant Physiol. 102: 1185-1192, 1993).
There is no report concerning functions or roles of RF0 in a plant body other than those in a seed. A seed and a plant body may share an overlapping mechanism for acquiring drought resistance, or they may have totally different mechanisms.
For example, it is known that stresses, such as drought conditions, cause a plant to close stomata by accumulation of ABA as described above to suppress transpiration, thereby preventing water loss. Actually, ABA-deficient Arabidopsis mutants aba1 having an altered ABA synthetic system wither easily such that they cannot grow at a normal humidity. However, ABA-deficient mutant seeds can bud even under completely drought conditions. In other words, no decrease in drought resistance is found in ABA-deficient mutant seeds (Koornneef, M et al., Physiol. Plant. 61: 377-383, 1984, Duckham, S. C. et al., Plant Cell and Environ. 14: 601-606, 1991, Rock, C. D. and Zeevaart, J. A. D., Proc. Natl. Acad. Sci. 88: 7496-7499, 1991).
Moreover, seeds of ABA insusceptible Arabidopsis mutant abi3 are known to lack drought resistance. Under complete drought conditions, the seeds lose their budding ability. However, the seeds disseminated before progression of drying can bud, and no phenotype that withers like aba1 is observed (Nambara, E., et al, Polant J. 2: 435-441, 1992, Kriz, A. R., et al, Plant Physiol. 92: 538-542, 1990, Parcy, F., et al, Plant Cell 6: 1567-1582). In conclusion, ABI3 possesses a mechanism to acquire drought resistance, which functions only in its seed, and whose action is far greater than of ABA.
As described above, there is a great difference between a seed and a plant body in respect of drought resistance acquisition mechanism. Whether RF0, which is suggested to be important in drought resistance of a seed, plays a role in drought resistance of a plant body remains unknown and cannot even be predicted.
Accordingly, an object of the present invention is to provide a method for increasing stress resistance to a plant, which enables production of a plant having resistance against environmental stresses including drought and/or high salt concentration.
After thorough study, the inventors have completed the invention by finding that an increased content of galactinol in a plant body can provide the plant with stress resistance.
The present invention encompasses the followings:
(1) a method for increasing stress resistance to a plant wherein a galactinol synthetase gene is introduced into the plant body.
(2) The method for increasing stress resistance to a plant according to (1) wherein the galactinol synthetase gene is the following gene (a) or (b):
(a) a gene encoding a protein comprising an amino acid sequence represented by SEQ ID NO: 1,
(b) a gene encoding a protein comprising an amino acid sequence differing from the amino acid sequence of SEQ ID NO: 1 by deletion, substitution or addition of at least one or more amino acids, and having galactinol synthetic activity.
(3) The method for increasing stress resistance to a plant according to (1) wherein the galactinol synthetase gene is the following gene (c) or (d):
(c) a gene encoding a protein comprising an amino acid sequence represented by SEQ ID NO: 2,
(d) a gene encoding a protein comprising an amino acid sequence differing from the amino acid sequence of SEQ ID NO: 2 by deletion, substitution or addition of at least one or more amino acids, and having galactinol synthetic activity.
(4) The method for increasing stress resistance to a plant according to (1) wherein galactinol content in the plant body is increased.
(5) The method for increasing stress resistance to a plant according (1) wherein galactinol synthetic activity in the plant body is improved.
(6) A method for increasing stress resistance to a plant which comprises increasing galactinol content in the plant body.
(7) A method for increasing stress resistance to a plant which improving galactinol synthetic activity in the plant body.
(8) A method for increasing stress resistance to a plant which excessively expressing the following protein (e) or (f) in the plant body:
(e) a protein comprising an amino acid sequence represented by SEQ ID NO: 1,
(f) a protein comprising an amino acid sequence differing from the amino acid sequence of SEQ ID NO: 1 by deletion, substitution or addition of at least one or more amino acids, and having galactinol synthetic activity.
(9) A method for increasing stress resistance to a plant which excessively expressing the following protein (g) or (h) in the plant body:
(g) a protein comprising an amino acid sequence represented by SEQ ID NO: 2,
(h) a protein comprising an amino acid sequence differing from the amino acid sequence of SEQ ID NO: 2 by deletion, substitution or addition of at least one or more amino acids, and having galactinol synthetic activity.